Dynamics And Mechanisms For Chronic Intermittent Hypoxia-induced Cardiac Damage And Its Protection By Metallothionein

Backgroud:Obstructive sleep apnea (OSA) is a highly prevalent respiratory disorder of sleep,characterized by recurrent episodes of complete or partial collapse of the upper airwayduring sleep associated with repetitive apneas or hypopneas. Both apneas and hypopneasevents can produce chronic intermittent hypoxia (CIH), sleep arousals also called sleepfragmentation (SF) and hemodynamic changes. Among these, CIH is the main reason ofOSA-induced pathological damage. OSA is considered as a life-threatening condition and asan independent risk factor of cardiovascular diseases. OSA has a direct correlation withmorbidity and mortality of cardiovascular diseases. Now, it has become the most importanttarget of public health intervention aiming at reducing cardiovascular diseases.Although OSA is related with several of cardiovascular diseases, its mechanisms arenot well known. Oxidative stress is considered as one of the major mechanisms of OSA-induced cardiovascular diseases. We hypothesis that cardiac response to CIH exists withtime-dependent manner. Metallothionein (MT) is a specific, effective, endogenous andpowerful antioxidant. It can express in several organs including heart. We get the hypothesisthat MT as an endogenous antioxidant can protect the heart against OSA-induced cardiacdamage. Now, there is no related report about the protection of MT against OSA-inducedcardiovascular diseases.Objectives:In our study, we used CIH exposure mice model to mimic human's condition of OSA.We want to elucidate the time-dependent manner of cardiac response to CIH by dynamicobservation on the mice exposed to CIH. We also want to confirm that oxidative stress is themain mechanism of CIH-induced cardiovascular diseases. At the same time, we used twokinds of antioxidant to treat the FVB mice and gave CIH in order to elucidate that oxidativestress is the major mechanism of OSA-induced cardiac damage. We also want to elucidatethe MT's protective effects against CIH-induced cardiac damage as an endogenousantioxidant by exposure of the cardiac specific MT overexpression transgene (MT-TG)mice to CIH in order to confirm the effects of oxidative stress in the CIH-induced cardiac damage. We also want to provide the theoretic basis of OSA-induced cardiovascular diseasesand also want to find new target of the therapy.Methods:Three sets of experiments were done: The first study was designed to investigate thetime course of cardiac responses to CIH from3days to4weeks using wild type FVB miceonly; The second study was designed to investigate the mechanism of CIH-induced cardiacdamage by using two kinds of antioxidant. The third part of the study was to investigate theprotective effect of cardiac MT on CIH-induced cardiac damage by exposure of both MT-TG mice and wild type mice for4and8weeks.1Cardiac response to CIH with a time-dependent mannerIn order to observe if cardiac response to CIH has a time-dependent manner andcomfirm its mechanism, we exposed8-10weeks old male wild type FVB mice to CIH for3days,7days (1week),14days (2week),21days (3weeks),28days (4weeks) and56days(8weeks) and detected the items below at different time point.(1) Body weight, heart weight and the ratio of heart weight to tibia length: We detectedbody weight, heart weight, tibia length and calculated the ratio of body weight to tibia lengthin order to observe if CIH exposure can influence the growth and induce cardiac hypertrophy.(2) Detection of cardiac hypertrophy marker gene: We detected the expression ofcardiac hypertrophy marker fetal gene atrial natriuretic peptide (ANP) and β-myosin heavychain (β-MHC)'s expression at mRNA level using real-time polymerase chain reaction(RT-PCR) and ANP's expression at protein level using Western-blot methods.(3) Detection of cardiac struction and function: We assessed cardiac structure andfunction using a Visual Sonics Vevo770high-resolution imaging system at different timepoint.(4) Detection of cardiac fibrosis: We detected cardiac fibrosis-related items includingconnective tissue growth factor (CTGF) and plasminogen activator inhibitor-1(PAI-1)'sexpression at protein level using Western-blot and detected the collagen accumulation withSirius-red staining using0.1%Sirius Red F3BA and0.25%Fast Green FCF.(5) Detection of cardiac inflammation: We detected inflammation-related itemsincluding intracellular adhesion molecule-1(ICAM-1) and vascular cell adhesion molecule-1(VCAM-1)'s expression at protein level using Western-blot methods and detected theinfiltration of neutrophil with Naphthol A.S-D Chloroacetate Esterase staining. (6) Detection of cardiac apoptosis: We detected cardiac apoptosis-related itemsincluding CIEBP homologous protein (CHOP) and cleaved-caspase-3(C-Cas-3)'sexpression at protein level using Western-blot and cardiac apoptotic cell death was measuredby terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assaywith the ApopTag Peroxidase In Situ Apoptosis Detection Kit.(7) Detection of cardiac oxidative stress: Malondialdehyde (MDA) production wasmeasured by thiobarbituric acid (TBA) assay as an index of lipid peroxidation. We alsodetected antioxidants such as MT, Cu/Zn superoxide dismutase (SOD1) and nuclear factor-erythroid2-related factor2(Nrf2)'s expression at protein level with Western-blot andconfirm the expression of MT's expression with immunohistochemistry method.2Mechanism of CIH-induced cardiac damageIn order to elucidate the mechanism of CIH-induced cardiac damage, we used two kindsof antioxidants: reduced form of nicotinamide-adenine dinucleotide phosphate oxidase(NOX) inhibitor Apocynin (3mg/Kg/day, intraperitoneal injection for4weeks) and SODmimic MnTMPyP(5mg/Kg/day, intraperitoneal injection for4weeks)to treat the FVBmice and gave CIH for4weeks. We evaluated cardiac response to CIH from general sample,cardiac structure and function and molecular levels.3Protection of MT against CIH-induced cardiac damageIn order to detect if MT as an endogenous antioxidant has a protective effect againstCIH-induced cardiac damage, we exposed8-10weeks male MT-TG mice and their age-matched wild type mice to CIH for4and8weeks to observe the response of the heart to CIH.We detected the items using the same methods as mentioned above.Results:1Cardiac response to CIH in wild type FVB mice has a time-dependent manner(1) Body weight: In control group, animals gained their body weight in an age-dependent manner from0day to8weeks. Exposure to CIH inhibited body-weight gain from3days to3weeks and8weeks but did not significantly influence it at the4weeks. Itsuggested that CIH could inhibit the increase of body weight at relative early and late stages.(2) Heart weight, tibia length and the ratio of heart weight to tibia length: There was nodifference for tibia length between control and CIH group at each time-point. CIH delayedthe heart growth relative to controls, shown by a low heart weight in CIH group compared tothat in control group at the early stage (1week), and gradually increased from2weeks, andsignificantly increased at the4weeks, reflected by the absolute heart weight and the ratio of heart weight to tibia length. These measurements indicate that CIH may induce cardiachypertrophy at4weeks.(3) ANP and β-MHC's expression: We detected cardiac hypertrophy marker genesANP and β-MHC's expression at mRNA and protein levels and found that expressions ofANP and β-MHC were increased significantly at the end of3weeks of CIH exposure andfurther increased significantly at4weeks. At8weeks, CIH remained slightly increase inANP and β-MHC mRNA expressions but not statistical difference and also less than theeffects observed in mice exposed to4weeks. These results confirmed that CIH inducedcardiac hypertrophy from3weeks to4weeks.(4) Cardiac function: Cardiac function was evaluated by echocardiography, whichrevealed that the heart seemed to be dilated at4weeks of CIH exposure, shown by anincrease in left ventricle caviar dimension (LVID) both at diastole and systole and decreasein cardiac systolic function at4weeks, shown by decreases in left ventricle ejection fraction(LVEF) and left ventricle fraction shortening (LVFS). Echocardiography analysis exhibitedsignificantly structural changes at8weeks, shown by the decrease of interventricle septemthickness (IVS) at diastole, left ventricle posterious wall thickness (LVPW) at both diastoleand systole, the increase of LVID at both diastole and systole, and further decreases in LVEFand LVFS. These results implied that at the early stage after exposure to CIH, the heartmight be compensative but at the relative late stage, the heart became enlargement, at whichcardiac dysfunction happens.(5) Cardiac fibrosis: Sirius-red staining for collagen accumulation showed thatsignificant positive staining was observed in the heart of mice exposed to CIH for4weeksand8weeks. Western-blot of PAI-1and CTGF also revealed the increased expression ofthese two fibrotic mediators at the4weeks and8weeks of CIH exposure. It suggested thatCIH could induce cardiac fibrosis from4weeks CIH exposure.(6) Cardiac inflammation: ICAM-1and VCAM-1's expression revealed that bothinflammatory cytokines were decreased in the early stages (3days and1week), butsignificantly elevated at the4weeks in CIH group. A significant increase in infiltration ofneutrophil was found only in the heart of mice exposed to CIH for4weeks. We also foundthat cardiac inflammation disappeared at8weeks.(7) Cardiac apoptosis: There was a significant increase in apoptotic cells in the heart ofmice exposed to CIH for4weeks and8weeks, which was confirmed by TUNEL stainingand by the protein expression of CHOP at4weeks and C-Cas-3at8weeks detected by Western-blot assay in heart tissue. It suggested that CIH could induce cardiac apoptosis since4weeks exposure.(8) Cardiac oxidative stress: Oxidative damage, examined by MDA, a marker of lipidperoxide, significantly increased at4and8weeks. As antioxidant, SOD1's expression wasnot changed during3days to4weeks, but decreased significantly at8weeks. MT'sexpression significantly increased at3days and returned to normal levels at1-2weeks anddecreased from3weeks to8weeks compared to control group. Nrf2's expressionsignificantly increased at3days, and returned to normal levels at1-3weeks and decreased at4and8weeks, compared to control group. These results suggest that oxidative stress is themain reason of CIH-induced cardiac damage and antioxidant up-regulation is the mainmechanism for cardiac compasative response and the downregulation of antioxidant'sexpression is the main reason for cardiac decompasitive response to CIH.2Protective effects of antioxidants on CIH-induced cardiac damageThere's no significant difference of body weight, tibia length, heart weight and the ratioof heart weitht to tibia length between control and CIH group treated with these two kinds ofantioxidants. There's also no significant difference about ANP, CTGF, PAI-1, VCAM-1andMDA's expression between these two groups. Compared to control group, there was also nosignificant differende about cardiac structure and function's changes. These resultssuggested that antioxidant can protect the heart against CIH-induced cardiac damage. It alsosuggested that oxidative stress was the main mechanism of CIH-induced cardiac damage.3Protective effects of MT on CIH-induced cardiac damage.MT-TG mice were exposed to CIH for4and8weeks. We found that at different timepoint, there was no change for body-weight gain, tibia length, heart weight, and the ratio ofheart weight to tibia length. There was no sign for cardiac hypertrophy and fibrosis byexamination of ANP mRNA and protein expression, CTGF protein expression and Sirius redstaining. MT-TG mice were resistant to CIH-induced cardiac apoptosis, inflammation, andcardiac oxidative stress. These results suggested that MT could protect the heart againstCIH-induced cardiac damage and MT has an import role for the transition of cardiacresponse from compasative stage to decompasative stage.Conclusions:1Cardiac response to CIH manifested a transition from adaptation to maladaptation i.e.heart began to have a transition from compensation to decompensation with a time-dependent manner. 2Oxidative stresses is the main mechanism of CIH-induced cardiac damage and thedecrease of antioxidant capacity is the main reason of oxidative stresses.3Expression of MT in heart tissues is the main compensatory mechanism for CIH-induced cardiac damage.4MT as an endogenous antioxidant has definite protective effects on the heart againstCIH-induced cardiac damage.Innovations:1We observed cardiac response to CIH dynamically and elucidated that CIH-inducedcardiac damage existed a time-dependent manner.2We elucidated further that oxidative stresses is the main mechanism of CIH-inducedcardiac damage and the decrease of antioxidant capacity was the main reason of oxidativestresses.3We also confirmed for the first time that MT's expression in the heart tissues was acompensatory mechanism of CIH-induced cardiac damage.4We observed the protective effects of MT and comfirmed the role of MT'sexpression in the process of CIH-induced cardiac damage for the first time.